Regulated overexpression of the survival factor bcl-2 in CHO cells increases viable cell density in batch culture and decreases DNA release in extended fixed-bed cultivation
Authors: Fussenegger M.1; Fassnacht D.2; Schwartz R.2; Zanghi J.A.1; Graf M.1; Bailey J.E.3; Pörtner R.2
Source: Cytotechnology, Volume 32, Number 1, 2000 , pp. 45-61(17)
Publisher: Springer
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Abstract:
Using multicistronic expression technology we generated a stable Chinese hamster ovary (CHO) cell line (MG12) expressing a model secreted heterologous glycoprotein, the secreted form of the human placental alkaline phosphatase (SEAP), and bcl-2, best known as an apoptosis inhibitor, in a tetracycline-repressible dicistronic configuration. In batch cultivations in serum-containing medium, MG12 cells reached twice the final viable cell density when Bcl-2 was overexpressed (in the absence of tetracycline) compared to MG12 populations cultured under tetracycline-containing conditions (bcl-2 repressed). However, bcl-2-expressing MG12 cells showed no significant retardation of the decline phase compared to batch cultures in which the dicistronic expression unit was repressed. Genetic linkage of bcl-2 expression with the reporter protein SEAP in our multicistronic construct allowed online monitoring of Bcl-2 expression over an extended, multistage fixed-bed bioreactor cultivation. The cloned multicistronic expression unit proved to be stable over a 100 day bioreactor run. CHO MG12 cells in the fixed-bed reactor showed a drastic decrease in the release of DNA into the culture supernatant under conditions of reduced tetracycline (and hence derepressed SEAP and bcl-2 overexpression). This observation indicated enhanced robustness associated with bcl-2 overexpression, similar to recent findings for constitutive Bcl-2-overexpressing hybridoma cells under the same bioprocess conditions. These findings indicate, in these serum-containing CHO cell cultures, that overexpression of Bcl-2 results in desirable modifications in culture physiology.
Keywords: apoptosis; Bcl-2; fixed-bed reactor; regulated gene expression
Language: English
Document Type: Regular paper
Affiliations: 1: Institute of Biotechnology, ETH Zürich, CH-8093 Zürich, Switzerland 2: Technische Universität Hamburg-Harburg, Biotechnologie 1, D-21071 Hamburg, Germany 3: Institute of Biotechnology, ETH Zürich, CH-8093 Zürich, Switzerland (Author for all correspondence)
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